This grant is directed at understanding the molecular mechanisms that control the development and connectivity of spinal neurons involved in the control of locomotion. Motor neurons represent one of the key cell types that directly mediate the control of movement and respiration by forming synaptic connections with muscles. Therefore diseases of these cells such as ALS, post polio syndrome, and spinal muscle atrophy are extremely debilitating and frequently lethal. Although tremendous progress has been made in identifying the factors that trigger motor neuron development, regulate gene expression, and mediate synapse formation, our understanding of the molecular mechanisms by which some of these factors act is in many cases rather limited. During development motor neuron subtypes are generated that exhibit distinct cell migration patterns and whose axons display specific preferences for particular nerve pathways. A family of LIM homeodomain (LIM-HD) transcription factors is expressed in unique combinations within individual motor neuron subtypes (LIM code). Moreover, functional experiments have now demonstrated that the combinatorial activity of LIM-HD factors contribute to the specification of distinct types of motor neurons as monitored by examining motor neuron cell migration, axon navigation, and gene profiles. Therefore LIM-HD factors are intimately involved in establishing motor neuron identity and connectivity. This proposal addresses two main deficiencies in our understanding of the LIM-HD factors: how they actually regulate gene expression, and the identity and function of the target genes under their control. In the first aim we plan to address the biochemical basis for the LIM code and to examine how LIM-HD factors act in the context of other regulatory factors such as those that control neurogenesis. In the second aim we will test the function of LIM-only factors to determine whether they negatively-regulate the activity of the LIMHD factors. In the third aim we plan to characterize the molecular basis for NLI's involvement with the LIM-HD factors. And in aim four we will screen for target genes of the LIM-HD factors and characterize their function. Over the long term these studies should contribute to our general understanding of the molecular mechanisms that control neuronal differentiation and provide insight into novel methods for restoring motor function lost due to injury or disease